Michihiro Takai

A potent and specific agonist, Suc-[Glu9, Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.

A novel subtype of endothelin B receptor mediating contraction in swine pulmonary vein.

Effects of agonists and antagonists of endothelin (ET) receptors were examined in swine pulmonary artery and vein and in rabbit saphenous vein. ET-1, but not ETB receptor agonists, sarafotoxin S6c (STXc) and IRL 1620, induced contraction in pulmonary artery. This effect was inhibited by the ETA receptor antagonists, BQ-123 and FR139317, but not by the ETB receptor antagonist, IRL 1038. Pulmonary artery precontracted by norepinephrine was relaxed by ET-3 in an endothelium-dependent manner. This relaxation was inhibited by IRL 1038 but not by BQ-123. In pulmonary vein, ET-1, ET-3, STXc and IRL 1620 induced contractions at a similar concentration range. ET-1 induced contraction also in saphenous vein. These contractions were not inhibited by BQ-123, FR139317 or IRL 1038. These results suggest that the isopeptide-selective ETA receptor mediates contraction in swine pulmonary artery whereas the isopeptide-nonselective ETB receptor mediates release of endothelium-derived relaxing factor. In contrast, contractions in the veins may be mediated by a novel subtype of isopeptide-nonselective ETB receptor which is not inhibited by IRL 1038.

An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11–21)

In the inhibition of specific binding or [125]endothelins (ETs) to membranes from various tissues of rats, guinea pigs, pigs and humans, [Cys11‐Cys15]‐ET‐1(11–21), IRL 1038, has a much higher affinity for ETB receptors (K i = 6–11 nM) than for ETA receptors (K i = 0.4–0.7 μM). In contraction assays, with ET‐3 as a stimulant, 3 μM IRL 1038 antagonized the ETB receptor‐mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor‐mediated contraction of rat aortic smooth muscle. IRL 1038 is, therefore, considered to be the first antagonist selective to the ETB receptor.

Induction of endothelium‐dependent relaxation in the rat aorta by IRL 1620, a novel and selective agonist at the endothelin ETB receptor

1 The effects of a novel and selective agonist at the endothelin ETB receptor, IRL 1620 (Suc‐[Glu9, Ala11,15] endothelin‐1 (8–21)), were examined in the isolated aorta of the rat. 2 IRL 1620 (1–300 nm) changed neither the resting tone nor the cytosolic Ca2+ level ([Ca2+]i) of the aorta without endothelium. In the presence of endothelium, however, IRL 1620 increased endothelial [Ca2+]i with little effect on the muscle tone. In the absence of external Ca2+, IRL 1620 still induced a transient increase in endothelial [Ca2+]i. 3 Noradrenaline (100 nm) increased both muscle [Ca2+]i and tension. IRL 1620 (1–300 nm) relaxed the muscle with an increase in endothelial [Ca2+]i only in the presence of endothelium. An inhibitor of nitric oxide synthase, 100 μm NG‐monomethyl‐l‐arginine, inhibited the relaxant effect of IRL 1620 but not the increase in endothelial [Ca2+]i. 4 In resting and noradrenaline‐stimulated aorta, the effects of IRL 1620 were inhibited by a selective antagonist of the ETB receptor, IRL 1038 (0.3–3 μm), although a selective antagonist of the ETA receptor, BQ‐123 (3 μm), was ineffective. Verapamil (10 μm) did not alter the effects of IRL 1620. 5 A muscarinic receptor agonist, carbachol (1 μm), also induced endothelium‐dependent relaxation with an increase in endothelial [Ca2+]i. However, the effects of carbachol were not inhibited by the ETB antagonist, IRL 1038 (3 μm). 6 These results suggest that IRL 1620 is a selective agonist at the ETB receptor which increases endothelial [Ca2+]i by releasing Ca2+ from storage sites and by opening non‐L type Ca2+ channels, activates nitric oxide synthase, releases nitric oxide, and relaxes vascular smooth muscle.

ETB receptor antagonist, IRL 1038, selectively inhibits the endothelin-induced endothelium-dependent vascular relaxation

In isolated rat aorta, endothelin-1 induced contractions at lower concentrations than endothelin-3. The contractile effects were augmented by removing the endothelium. In contrast, endothelium-1 and endothelin-3 at similar concentrations induced endothelium-dependent relaxation in norepinephrine-stimulated aorta. IRL 1038 ([Cys11,Cys15]endothelin-1(11-21); 3 microM) augmented the contractile effects of endothelins only in the presence of the endothelium. IRL 1038 (0.3-3 microM) inhibited the endothelium-dependent relaxation induced by endothelins but not by carbachol. IRL 1038 itself did not change muscle tension. These results suggest that IRL 1038 is a novel antagonist of the ETB receptor responsible for the release of relaxing factor from the vascular endothelium.

Renal vasodilating and diuretic actions of a selective endothelin ETB receptor agonist, IRL1620

The effects of a selective agonist for endothelin ETB receptors, Suc-[Glu9,Ala11,15]endothelin-1-(8-21), IRL1620, on renal hemodynamics and urine formation were studied in anesthetized dogs. Intrarenal arterial infusion of IRL1620 at a dose of 50 ng/kg/min increased renal blood flow from 3.37 +/- 0.30 (mean +/- S.E.) to a maximal value of 4.43 +/- 0.45 ml/g kidney weight per min (ml/g/min) at 9.1 +/- 1.0 min after the start of infusion, with no change in systemic blood pressure and heart rate. Urine flow rate increased and urine osmolality, osmolar clearance and free water reabsorption decreased significantly whereas glomerular filtration rate remained unchanged. In dogs given ibuprofen (12.5 mg/kg, i.v.) after the start of infusion of the peptide, renal blood flow increased slightly but significantly from 3.78 +/- 0.82 to 4.17 +/- 0.96 ml/g/min (1.0 +/- 0.1 min), followed by a gradual reduction in renal blood flow. In dogs given L-NG-nitroarginine (75 micrograms/kg/min), the renal blood flow decreased following intrarenal administration of IRL1620 (50 ng/kg/min). It is suggested that IRL1620 enhances the release of nitric oxide and prostaglandins in the kidney and promotes renal vasodilation. The IRL1620-induced reduction of urine osmolality and free water reabsorption was affected by neither ibuprofen nor L-NG-nitroarginine, thereby suggesting that the suppression of urine concentration did not seem to be linked to the enhanced production of nitric oxide or prostaglandins in the kidney.

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.

Retraction concerning an endothelin B receptor-selective antagonist: Urade, Y., Fujitani, Y., Oda, K., Watakabe, T., Umemura, L., Takai, M., Okada, T., Sakata, K. and Karaki, H. (1992) FEBS Lett. 311, 12-16 : An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11-21)

In the inhibition of specific binding of [125I]endothelins (ETs) to membrane from various tissues of rats, guinea pigs, pigs and humans, [Cys11-Cys15]-ET-1(11-21), IRL 1038, has a much higher affinity for ETB receptors (Ki = 6-11 nM) than for ETA receptors (Ki = 0.4-0.7 microM). In contraction assays, with ET-3 as a stimulant, 3 microM IRL 1038 antagonized the ETB receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is therefore, considered to be the first antagonist selective to the ETB receptor.The article describes the synthetic peptide IRL 1038 as having a much higher affinity to ETB receptors (Ki = 6 11 nM) than to ET, receptors (Ki = 4OC700 nM) in various tissue membranes. It was also shown that this peptide antagonized the ET,-mediated contraction of guinea pig ileal and tracheal smooth muscles. Since the publication of the data, we have synthesized more than 20 different batches of IRL 1038, some of which showed potencies and selectivities almost equivalent to the published data. However, the other batches, including batches synthesized by external suppliers, exhibited significantly higher Ki of 5&400 nM for the binding to ET, receptors in porcine lung membranes. We have very carefully investigated the physico-chemical properties of IRL 1038 in aqueous solutions at different pH (pH 7-10) and peptide concentrations, using fluorescence and proton NMR spectroscopy. At high pH (pH lo), the peptide does not aggregate and two kinds of non-aggregated states were observed by NMR spectroscopy. There was, however, no evident correlation between the two non-aggregated states and binding af-

Solution synthesis of [ASN76]-human parathyroid hormone(1-84)

Human parathyroid hormone, hPTH(1-84), was synthesized by the conventional solution procedure applying the maximal-protection approach. All protecting groups were removed simultaneously by the HF method. The product was purified by CM-cellulose column chromatography, gel-filtration on Sephadex G-50 and in the final stage, by reversed phase HPLC. The structure of the final product was confirmed not only by HPLC analysis but also by peptide mapping of tryptic digests on HPLC. The present product showed 350(249-480) IU/mg on in vitro rat renal adenylate cyclase assay.

Conformational Studies on the Specific Cleavage Site of Type I Collagen (α-1) Fragment (157–192) by Cathepsins K and L by Proton NMR Spectroscopy

Cathepsins K and L are cysteine proteinases which are considered to play an important role in bone resorption. Type I collagen is the most abundant component of the extracellular matrix of bone and regarded as an endogenous substrate for the cysteine proteinases in osteoclastic bone resorption. We have synthesized a fragment of Type I collagen (alpha-1) (157-192) as a substrate for the cathepsins and found that cathepsins K and L cleave the fragment at different specific sites. The major cleavage sites for cathepsin K were Met159-Gly160, Ser162-Gly163 and Arg165-Gly166, while those for cathepsin L were Gly166-Leu167 and Gln180-Gly181. The structure of the fragment was analyzed in aqueous solution by circular dichroism and proton NMR spectroscopy and the difference in the molecular recognition of collagen by cathepsins K and L was discussed from the structural aspect.